1. Field of the Invention
The present invention is directed to improved processes and compositions for the isolation of proteins, and to novel genetic constructions allowing the ready isolation of desired proteins or peptides, particularly multi-chain proteins such as human relaxin that are essentially devoid of aspartic acid ("Asp") residues.
2. Description of Related Disclosures
The production and isolation of desired proteins by recombinant techniques, for example, employing genetically engineered or isolated gene sequences, has in recent years reached a moderate level of sophistication. In fact, it is now possible to produce a variety of proteins by recombinant techniques, including, for example, recombinant human interferon, human growth hormone, or human tissue plasminogen activator, to name just a few, in a variety of hosts, including both eukaryotic and prokaryotic hosts [Maniatis et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor: New York, 1982)]. Moreover, techniques for moving or "engineering" DNA sequences from one context to another, for example, translocation of sequences from one recombinant vector or host to another vector or host, is currently achievable on a routine basis. Such successes have allowed the production and ready availability of a number of important pharmaceutical and biotechnical products, in a form essentially free of materials normally associated with the protein in its natural environment.
Unfortunately, certain proteins are expressed by recombinant means only with some difficulty. For example, certain proteins, and in particular certain protein hormones, naturally exist in a mature form quite distinct from their cellular nascent form, requiring processing, often the action of a series of enzymes. Such proteins are said to exist in pre-, pro-, or pre/pro-forms. Processing of such proteins will also often result in the generation of two or more individual peptide chains, one or both of which may have biological activity, or which may themselves form bonds or crosslinks resulting in multi-chain proteins, e.g., insulin or relaxin.
The principal problem encountered in generating such proteins is the requirement that pre- and post-sequences, or internally located sequences, be somehow removed to provide the mature protein. Under certain circumstances, such a problem has been reduced or minimized through the use of eukaryotic expression systems wherein the expressed protein or peptide is adequately processed by the eukaryotic host. Unfortunately, such in vivo processing is not always entirely faithful. When this is the case, one is left with a pre- or pro-protein material, often exhibiting only slight or low intrinsic levels of biological activity. Without a convenient means of further processing these proteins, they are only of minimal or no use medically or otherwise. Moreover, in certain instances it is preferable to produce recombinant expression products in a prokaryotic host, such as a bacterium, wherein much larger quantities of expression product may at times be produced more economically.
An example of a protein that ordinarily must be post-translationally modified, e.g., into separate protein chains, is human relaxin. Relaxin is a mammalian peptide hormone that plays an important role in facilitating the birth process through its effects in dilating the pubic symphysis [see, e.g., Hisaw, Proc. Soc. Exp. Biol. Med., 23: 661 (1926)]. Relaxin is synthesized and stored in the corpora lutea of ovaries during pregnancy and is released into the blood stream prior to parturition. Its primary physiological actions appear to be involved in preparing the female reproductive tract for parturition. These actions include dilation and softening of the cervix, inhibition of uterine contractions, and relaxation of the pubic symphysis and other pelvic joints.
The availability of ovaries from pregnant animals has enabled the isolation and amino acid sequence determination of relaxin from pig [see, e.g., Schwabe et al., Biochem. Biophys. Res. Comm., 75: 503-510 (1977); James et al., Nature, 267: 544-546 (1977)], rat [John et al., Endocrinology, 108: 726-729 (1981)], and even shark [Schwabe et al., Rec. Progr. Horm. Res., 34: 123-211 (1978)]. Moreover, recombinant DNA techniques have allowed the cloning and expression of various relaxins, including, in particular, porcine relaxin [see EPO Pub. No. 86,649] and human relaxin [see, e.g., EPO Pub. No. 101,309 and U.S. Pat. No. 2,758,516, the disclosures of which are incorporated herein by reference].
From the foregoing and other work, it is now known that the relaxin molecule, including both its initial translation transcript (prepro relaxin) and processed mature form (relaxin), bear a striking resemblance to corresponding forms of insulin. For example, relaxin is originally translated in a "prepro" form that bears a prehormone sequence (thought to play a role in extrusion and possibly folding of the peptide in the endoplasmic reticulum) and a prohormone sequence comprising three regions, the so-called B, C, and A chain-coding regions (generally arrayed in that order). Post-translational processing of preprorelaxin to form mature relaxin involves the enzymatic cleavage, in its natural cellular environment, of pre- and C-region peptides to leave the B and A chain peptides, joined by disulfide bonds through cysteine residues, as well as an intra-chain disulfide bridge within the A-chain itself.
In man, relaxin is only found in one of two potential forms, designated herein as the Asp.sub.1 or H2 (human 2) and Lys.sub.1 or H1 (human 1) forms, corresponding to the two potential gene products in the human genome. In both forms, the A chain is devoid of Asp residues. However, in the H2 form, the relaxin B chain includes one Asp residue at position 1, whereas in the H1 form, the relaxin B chain includes Asp residues at positions 4 and 5.
There has existed a need for compositions and processes particularly adapted for the isolation of recombinant proteins that must be extensively processed through the removal of terminal and/or central peptides.
Fusion polypeptides have been prepared from appropriate microbial cloning systems that contain a methionyl residue at the fusion juncture for cleavage by cyanogen bromide. See, e.g., U.S. Pat. No. 4,356,270 issued Oct. 26, 1982. Moreover, linkers have been devised that code for an amino acid sequence representing a specific cleavage site of a proteolytic enzyme for cleavage of fusion proteins. See, e.g., U.S. Pat. No. 4,769,326 issued Sep. 6, 1988. Such processes provide recombinant technology with alternatives to eukaryotic cell expression. Further, it is known that a preferential hydrolysis of the peptide bonds of aspartyl residues occurs in dilute acid, resulting in cleavage of the peptide chain [see, e.g., Light, Meth. Enz. Vol. XI, p. 417-420 (1967); Ingram, Meth. Enz., Vol. VI, p. 831-834 (1963); Inglis et al. in Methods in Peptide and Protein Sequence Analysis, Birr, ed. (New York: Elsevier/North Holland Biomedical Press, 1980), pp. 329-343; Inglis, Meth. Enz., 91, 324-332 (1983); Schroeder et al., Biochemistry, 2: 992-1008 (1963) (p. 1005, left column, in particular); and Schultz, Meth. Enz., Vol. XI, p. 255-263 (1967)], and that preferential cleavage of aspartyl-prolyl peptide bonds takes place in dilute acid [see Marcus, Int. J. Peptide Proteins Res., 25: 542-546 (1985); Piszkiewicz et al., Biochem. Biophys. Res. Comm., 40: 1173-1178 (1970); Jauregui-Adell and Marti, Anal. Biochem., 69: 468-473 (1975); Landon, Meth. Enz., 47: 145-149 (1977)]. The Jauregui-Adell article suggests cleaving the Asp-Pro bond in the presence of strong denaturing agents to obtain reasonable yields. The Landon review article discloses that the use of guanidinium chloride is necessary to increase yields for one protein but not for another. The Inglis et al. article on p. 338 suggests that variations in amino acid sequence and environment surrounding the aspartic acid residues might affect the cleavage yields. For a thorough review of all nonenzymatic methods for preferential and selective cleavage and modification of proteins, see Witkop, in Advances in Protein Chemistry, Anfinsen et al., ed., Vol. 16 (Academic Press, New York, 1961), pp. 221-321, especially pp. 229-232 on aspartic acid cleavage.
UK 2,142,033 discloses cleavage of a fusion protein of IGF-I and Protein A by dilute acid treatment of a variant of the fusion protein having an Asp residue engineered at the proper fusion junction.
Despite this knowledge, there still exists a need for improved methods to produce and isolate recombinant proteins, particularly those that must be extensively processed by removal of central and terminal peptides, in high yield, and to provide for the restructuring of recombinant products into more desirable forms, for example, for the production of larger quantities of peptides having more desirable structures for expression purposes.
In recognition of these needs, it is a general object of the present invention to provide improved recombinant processes and compositions for the production of protein- or peptide-encoding DNA sequences.
It is an additional object of the present invention to provide improved processes for the production of desired proteins employing genetically engineered compositions.
It is a more particular object of the present invention to provide improved processes for providing recombinant relaxin, and in particular, human relaxin.